A lateral flow assay for the immunodiagnosis of human cat‐transmitted sporotrichosis

Abstract Background Cat‐transmitted sporotrichosis (CTS) caused by Sporothrix brasiliensis has emerged as an important zoonosis in Brazil and neighbouring countries. Objectives Evaluate the performance of a lateral flow assay (LFA) for the detection of anti‐Sporothrix antibodies in human sera. Methods A LFA for the detection of anti‐Sporothrix antibodies (Anti‐Sporo LFA) in human sera, developed by IMMY, was evaluated using 300 human sera collected prospectively at the Hospital de Clínicas, Federal University of Paraná (HC‐UFPR), in Curitiba, Brazil. These specimens included 100 sera from patients with CTS. CTS cases were classified as follows: 59 lymphocutaneous, 27 fixed cutaneous,13 ocular, and one mixed form. One‐hundred specimens from patients with other mycoses, including cryptococcosis (n = 32), candidemia (n = 27), paracoccidioidomycosis (n = 14), aspergillosis (n = 10), histoplasmosis (n = 9), fusariosis (n = 4), lobomycosis (n = 1), chromoblastomycosis (n = 1), mucormycosis (n = 1) and trichosporonosis (n = 1). And 100 specimens from apparently healthy volunteers (AHV). Results The Anti‐Sporo LFA showed a global sensitivity of 83% (95% confidence interval [CI] = 74%–90%), a global specificity of 82% (95% CI = 76%–87%), and accuracy of 82% (95% CI = 77%–86%). By clinical form sensitivity was as follows: Mixed form 100%, ocular 92%, lymphocutaneous 83% and fixed cutaneous 78%. False‐positive results were observed in 11 specimens from people with other mycoses and 26 specimens from AHV. Conclusion and discussion This study presents the results of the evaluation of the first lateral flow assay for the detection of anti‐Sporothrix antibodies in human sera. The findings here show evidence that IMMY's Anti‐Sporo LFA is a promising tool for the rapid diagnosis of CTS.


Sporotrichosis is a neglected implantation mycosis caused by
Sporothrix spp. This is a disease with global distribution, but it is more frequently reported in tropical and subtropical regions around the world. [1][2][3][4][5] Sporotrichosis has an estimated global annual incidence rate of more than 40,000 cases per year, mainly located in endemic areas, but the data are limited due to the lack of mandatory reporting of this disease in most of the affected countries. 6 Since the late 1990s, an outbreak of cat-transmitted sporotrichosis (CTS) emerged initially in the state of Rio de Janeiro, Brazil. Then, in 2007, a new species, S. brasiliensis, was identified using molecular analysis. 1,[7][8][9] This species has the capacity of zoonotic transmission and emerged as an agent of cat-transmitted sporotrichosis (CTS). 1,7,8 In the last two decades, S. brasiliensis has spread, causing CTS outbreaks across the Brazilian territory and in recent years, sporadic cases in Argentina, and Paraguay. 2,5,8,[10][11][12][13][14][15][16] A sick domestic cat (Felis catus) transmits the yeast form of S. brasiliensis to other cats, humans, and dogs. Infection occurs by traumatic implantation largely from scratching and biting, and in some cases, by cutaneous or mucosal implantation by contact with skin exudates and respiratory secretions from sick cats. 1,2 Sporothrix brasiliensis is more virulent than other species of the Sporothrix genus, and the fixed lymphocutaneous and cutaneous forms are the most frequent clinical forms. However, CTS epidemic atypical clinical forms have been described as ocular, antigen hyperreactivity, osteoarticular, meningitis and other extracutaneous infections, adding challenges to the diagnosis of this disease. [1][2][3]5,7,[17][18][19] Due to the broad clinical spectrum of this disease, the differential diagnosis of sporotrichosis with other infectious and non-infectious diseases must be carried out, including tegumentary leishmaniasis, tuberculosis, pyoderma, cat scratch disease, chromoblastomycosis, phaeohyphomycosis and mycetoma. 5 Due to the low specificity of the clinical manifestation, the use of specific laboratory assays is key. 1,20 To control the zoonotic spread of this disease, the implementation of the One Health approach is needed. This approach integrates human, animal and environmental professionals such as microbiologists, veterinarians, physicians, epidemiologists and surveillance officers. 8,16,21 One of the key aspects of human and animal patients with zoonotic sporotrichosis is its early detection. 16,22 In addition, due to the increase of CTS with atypical manifestations, it is necessary to implement rapid and accurate testing. 1,2,16,18 The standard method for the diagnosis of sporotrichosis is the isolation of the fungus by culture, but this method is timeconsuming, requires well-trained professionals, and has variable sensitivity. 1,20,23,24 Direct examination and histopathological study have low sensitivity; less than 30%. 8,20 Intradermal reactivity using sporotrichin is not commercially available and its use is limited to some few highly specialised medical centres. 20,23 For the immune diagnosis of sporotrichosis, there is a commercially available latex agglutination system for antibody (Ab) detection, but its analytical performance varies according to the clinical form, ranging from 100% sensitivity for the diagnosis of disseminated forms, to 56% for the diagnosis of cutaneous disease. 8,20 Some in-house enzyme immunoassays (EIA) have been developed, showing high sensitivity and specificity, but these assays are limited to just a few laboratories. 8,23,24 CTS caused by Sporothrix brasiliensis is an emerging health problem in Brazil and a threat to bordering countries. 1,7,8 The aim of this study was to evaluate the performance of a lateral flow assay (LFA) for the rapid detection of anti-Sporothrix antibodies in human sera.

| Study design
A prospective cross-sectional study was done. Sera specimens were Patients with more than 30 days of CTS treatment were excluded, and patients classified as possible CTS were also excluded. All study participants were enrolled in the study after accepting their participation by signing the informed consent.
CTS was defined following the guidelines of the Brazilian ministry of health (Appendix A). 24,25 Mycological studies were done with those patients where access to specimens was possible. All CTS cases were treated and followed during their time at the HC-UFPR. Cases were not enrolled if they had been treated for CTS for 31 days or more. 25

| Specimens
A total of 300 human sera specimens were tested. Specimens were classified into the following three groups. Group #1: 100 sera from patients with proven (n = 37) or probable (n = 63) CTS. By CTS clinical form, we tested 59 patients with lymphocutaneous CTS, 27 patients with fixed cutaneous CTS, 13 patients with ocular CTS, and one patient with mixed form of CTS (osteoarticular and fixed cutaneous).
Group #2: 100 sera from patients with other mycoses. This group includes 32 patients with cryptococcosis, 27 patients with candidemia, 14 patients with paracoccidioidomycosis, 10 patients with aspergillosis, nine patients with histoplasmosis, four patients with fusariosis, one patient with lobomycosis, one patient with chromoblastomycosis, one patient with mucormycosis and one patient with trichosporonosis.
Group #3 corresponded to 100 sera from apparently healthy volunteers (AHV) without contact with sick cats or any lesions ( Figure 1).

| Specimen preparation and testing
First, sera specimens were diluted 1:441 using the kit specimen diluent. 100 μl of the diluted sera was then dispensed into a flat bottom tube/well, followed by the LFA strip. The assay was incubated at

| Statistical analysis
Calculation of the analytical performance of the test was done using 2 × 2 tables comparing the Anti-Sporo LFA against CTS diagnosis.
Using this table, the tests sensitivity, specificity, accuracy, positive and negative predictive values, and their respective 95% confidence intervals (CI) were calculated. 26 Categorical variables were compared using a chi-square test with significance level of 5%. Analyses were conducted using MedCalc software.

| RE SULTS
All CTS cases and patients from the control group came from the Curitiba metropolitan area, at the Parana state in Brazil. Among the group of patients with CTS, 116 subjects with sporotrichosis were identified. Of these, two were excluded due to sapronotic infection. Another 10 patients were excluded because CTS was classified as possible. Additionally, four individuals were excluded because they were on antifungal treatment for more than 30 days ( Figure 1). We observed a higher prevalence of CTS in women, 1.6:1 sex ratio (62 women and 38 men). In this study, we found 16% prevalence of childhood CTS. All proven CTS were confirmed by molecular identification of S. brasiliensis. In the group of 63 probable CTS, five patients were tested by microscopy and culture. All five patients tested negative with both tests. All patients with a diagnosis of CTS received systemic antifungal treatment with itraconazole, terbinafine or both medicines. All cases were cured by the end of treatment.
We observed that the sensitivity in children was 88%, and in adults, it was 84%. These were higher in comparison with the group of patients older than 60 years, 75% sensitivity, but these differences were not statically significant (p = 0.610) ( Table 4). We observed statistically significant differences (p = <0.001) in the sensitivity of the Anti-Sporo LFA when disease onset was more than 21 days (91%), in comparison with patients with early disease, less than 21 days (31%) ( Table 4).
False-positive results were found in 37 out of the 200 (18%) non-CTS specimens. Among these false-positive results, 11 were from the group of patients with other mycoses (Group #2).
We observed that the false positives were cross-reactions with specimens from patients with histoplasmosis (2 out of 10, 20% cross-reactivity), candidemia (4 out of 27, 15% cross-reactivity), paracoccidioidomyocis (2 out of 14, 14% cross-reactivity), cryptococcosis (2 out of 32, 6% cross-reactivity) and trichosporonosis (1 out of 1, 100% cross-reactivity) ( Tables 5,6). The other 26 falsepositive results were observed in specimens from AHV (Group #3). All AHV declared not having comorbidities, contact with sick cats suspected of CTS, or having any previous presentation of symptoms suggestive of sporotrichosis. These subjects were individuals with ages ranging from 14 to 65 years old and were students or healthcare workers. All were residents in the endemic region for CTS. LFA bands in the test zone observed in Group #3 were less intense than bands observed from specimens in Groups #1 and #2.

| DISCUSS ION
This report describes the development and evaluation of the performance of the first lateral flow assay for the detection of anti- Factors that can also affect sensitivity of antibody detection assays include host baseline conditions, such as patient's immunological status, age, the time of disease onset, the clinical form of the disease, fungal burden and exposure to antifungal treatment. 5,27,[30][31][32][33] We found a statistically significant correlation between assay sensitivity and time of disease onset; being higher sensitivity in patients with more than 21 days of symptoms.
Experimental studies in mice have demonstrated that detection of IgG antibodies appears after 14 days of infection, and in humans, it has already been observed that this can be longer than 21 days. 27,32 Ageing could be the factor that affects assay sensitivity. 24,31,34 In this study, we observed a lower frequency of false negatives in children (12%) compared with adults (18%), but this was not statistically significant. To increase the chance of detecting cases, it is recommended to evaluate acute and convalescent specimens.
This recommendation is based on the evaluation of seroconversion, which could add important evidence for the diagnosis of the disease. 35 Since several factors influence the performance of Ab detection assays, results must be carefully analysed and compared with the clinical and epidemiological data.
Among the CTS false-negative results, ten were lymphocutaneous CTS, six were fixed cutaneous CTS, and one was an ocular form. It is known that antibody circulation and immunoglobulin type could vary based on the clinical manifestation of the disease. In fixed cutaneous and lymphocutaneous forms, low concentrations of IgM and IgA have been reported. 5,27,30 Therefore, negative results cannot exclude the presence of CTS. 31 In addition, we observed two false-negative results in patients with comorbidities. As previously reported, it is well known that the immune status of the host can affect the production of antibodies, affecting the performance of antibody detection assays. 17 The Anti-Sporo antibody LFA presented a global specificity of 82%. In the group of specimens from patients with other mycosis, we observed 11% cross-reactions, this type of cross-reaction with other fungal infections has been previously reported using other antibody detection assays. 31,32,36,37 Studies using an EIA anti-Sporothrix de- Additionally, most of these volunteers were healthcare workers residing in the endemic region for sporotrichosis. These characteristics would increase the risk of exposure to Sporothrix and other fungal pathogens able to produce cross-reactions. 8,19,24 Based on these findings, it is recommended to perform this test only in people who are symptomatic, or with a strong epidemiological link to CTS. In addition, future investigations focusing on product improvement are needed.
These studies could be focused on the optimisation of antigen used for antibody detection, and the implementation of a LFA reader.